U.S. patent number 11,325,898 [Application Number 17/112,980] was granted by the patent office on 2022-05-10 for small molecule inducers of autophagy.
This patent grant is currently assigned to Board of Regents, The University of Texas System. The grantee listed for this patent is Board of Regents, The University of Texas System. Invention is credited to Jef De Brabander, Wei-Chung Chiang, Beth Levine, Qiren Liang.
United States Patent |
11,325,898 |
Brabander , et al. |
May 10, 2022 |
Small molecule inducers of autophagy
Abstract
Small molecule disruptors of Beclin-1/Bcl-2 protein-protein
interactions induce autophagy and hence are useful for treating a
variety of indications where stimulation of autophagy is
therapeutically useful, including cancer, infection immunity,
neurodegeneration, longevity.
Inventors: |
Brabander; Jef De (Dallas,
TX), Liang; Qiren (Dallas, TX), Levine; Beth (Dallas,
TX), Chiang; Wei-Chung (Dallas, TX) |
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents, The University of Texas System |
Austin |
TX |
US |
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Assignee: |
Board of Regents, The University of
Texas System (Austin, TX)
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Family
ID: |
1000006298048 |
Appl.
No.: |
17/112,980 |
Filed: |
December 4, 2020 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20210115024 A1 |
Apr 22, 2021 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/US2019/035099 |
Jun 2, 2019 |
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62680578 |
Jun 4, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D
405/06 (20130101); C07D 231/12 (20130101); C07D
413/04 (20130101); C07D 409/04 (20130101); C07D
403/04 (20130101); C07D 405/14 (20130101); C07D
401/04 (20130101); C07D 413/10 (20130101); C07D
405/12 (20130101); C07D 231/06 (20130101); C07D
405/04 (20130101); C07D 417/04 (20130101) |
Current International
Class: |
C07D
405/04 (20060101); C07D 409/04 (20060101); C07D
405/06 (20060101); C07D 417/04 (20060101); C07D
405/12 (20060101); C07D 405/14 (20060101); C07D
231/06 (20060101); C07D 403/04 (20060101); C07D
231/12 (20060101); C07D 401/04 (20060101); C07D
413/10 (20060101); C07D 413/04 (20060101) |
Field of
Search: |
;514/227.8 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Vajda; Kristin A
Attorney, Agent or Firm: Osman; Richard Aron
Government Interests
This invention was made with government support under Grant Number
U19-AI109725 awarded by the National Institutes of Health (NIH).
The government has certain rights in the invention.
Claims
What is claimed is:
1. A method of selectively inducing autophagy, comprising treating
a person in need thereof with a pharmaceutical composition
comprising a compound of structure I: ##STR00113## wherein R1 is 2-
or 3-furanyl or phenyl, or 2-, 3- or 4-pyridine; R2 is a 1 para
substituent selected from halogen, NO.sub.2, N, C1-C4 alkyl, C1-C4
alkoxy, C1-C4 carbonyl, C1-C4 carboxyl, C1-C4 cyano, C1-C4
sulfinyl, and C1-C4 sulfonyl, each optionally fluorinated; R3 is 0
substituents; R4 is H; and R5 is SO.sub.2R or CO.sub.2R, wherein R
is C1-C4 alkyl, optionally fluorinated; or a salt, hydrate, or
stereoisomer thereof.
2. The method of claim 1, wherein R1 is 2- or 3-furanyl.
3. The method of claim 1, wherein R1 is phenyl.
4. The method of claim 1, wherein R1 is 2-, 3- or 4-pyridine.
5. The method of claim 1, wherein R2 is a 1 para substituent that
is halogen.
6. The method of claim 1, wherein R2 is a 1 para substituent that
is NO.sub.2.
7. The method of claim 1, wherein R2 is a 1 para substituent that
is N.
8. The method of claim 1, wherein R2 is a 1 para substituent that
is C1-C4 alkyl, optionally fluorinated.
9. The method of claim 1, wherein R2 is a 1 para substituent that
is C1-C4 alkoxy, optionally fluorinated.
10. The method of claim 1, wherein R2 is a 1 para substituent that
is --C4 carbonyl, optionally fluorinated.
11. The method of claim 1, wherein R2 is a 1 para substituent that
is C1-C4 carboxyl, optionally fluorinated.
12. The method of claim 1, wherein R2 is a 1 para substituent that
is C1-C4 cyano, optionally fluorinated.
13. The method of claim 1, wherein R2 is a 1 para substituent that
is C1-C4 sulfinyl, optionally fluorinated.
14. The method of claim 1, wherein R2 is a 1 para substituent that
is C1-C4 sulfonyl, optionally fluorinated.
15. The method of claim 1, wherein R5 is SO.sub.2R, wherein R is
C1-C4 alkyl, optionally fluorinated.
16. The method of claim 1, wherein R5 is CO.sub.2R, wherein R is
C1-C4 alkyl, optionally fluorinated.
17. The method of claim 1, wherein the compound is selected from:
##STR00114## ##STR00115## ##STR00116## ##STR00117## ##STR00118##
##STR00119## ##STR00120## ##STR00121##
18. The method of claim 1 wherein the person is in need of
treatment for diabetes, infection, cancer, neurodegenerative
disease or aging.
19. The method of claim 1 wherein the person is in need of
treatment for cancer.
20. The method of claim 1 wherein the person is in need of
treatment for Zika virus infection.
Description
INTRODUCTION
Macroautophagy (hereafter referred to as autophagy) is a catabolic
pathway by which cells sequester unwanted or damaged cellular
proteins or organelles through a double membraned structure called
the autophagosome. This process is mediated by a set of
evolutionarily conserved genes, the autophagy-related (AIG)
genes,.sup.1-2 which function in nucleation of the autophagosomal
membrane, elongation of the autophagic membrane, sequestration of
cytoplasmic constituents, closure of the double membrane, fusion
with the lysosome, and degradation of the sequestered contents.
Autophagy plays significant physiological roles in cellular
survival and stress adaptation,.sup.3 metabolism,.sup.4
development,.sup.5-6 immunity,.sup.7 protein and organellar
homeostasis,.sup.8 and protection against aging..sup.9 Moreover,
several lines of evidence indicate a link between autophagy and
mammalian diseases including diabetes, infection, cancer,
neurodegenerative diseases, and aging..sup.3, 9-10 Whole-body or
tissue-specific genetic disruption of autophagy in mice leads to
multiple pathologies, including tissue abnormalities, aberrant
inflammation, impaired immunity, neurodegeneration, and
susceptibility to tumorigenesis..sup.11 In humans, mutations or
polymorphisms in autophagy genes are associated with susceptibility
to infection, cancer, inflammatory diseases, asthma, cerebral
palsy, frontotemporal dementia, amyotrophic lateral sclerosis
(ALS), Huntington's disease, and Parkinson's disease..sup.11-14
Moreover, gain-of-function mutations or enforced expression of
autophagy genes in mice have beneficial effects including improved
metabolism and tissue function, extended lifespan, neuroprotection,
and decreased tumorigenesis..sup.11, 15-19 Thus, the development of
autophagy-inducing agents provide a therapeutic approach to prevent
and/or treat certain diseases in clinical medicine..sup.11,
20-22
Several drugs that are currently in clinical trials or clinical use
are known to induce autophagy.sup.11, 23; however, the effects of
these drugs are pleiotropic and their actions are not limited to
the autophagy pathway. Specifically, many drugs enhance autophagy
through the modulation of upstream signaling pathways (such as mTOR
inhibition, AMPK activation, calcium channel inhibition, and cAMP
signaling) but they also regulate diverse downstream biological
functions, thereby resulting in non-autophagy-related effects that
may limit clinical utility..sup.21 Therefore, to maximize benefits
and minimize toxicity, there is an urgent need for
autophagy-inducing agents that selectively target rate-limiting
steps in autophagy execution rather than upstream signaling.
One of the key mechanisms that regulates autophagy initiation is
the binding of Bcl-2 to Beclin 1, a scaffold protein that is an
essential determinant of the activity of the Beclin 1-VPS34 class
III phosphatidylinisotol 3 kinase complex..sup.24-25 During basal
conditions, autophagy levels are constrained by the binding of
Bcl-2 (or its related family member, Bcl-xL) to Beclin 1. In
response to stressful stimuli (such as nutrient starvation,.sup.24,
26 JNK activation,.sup.26 ceramide,.sup.27 or immunological
signaling.sup.28), the disruption of the Beclin 1/Bcl-2 complex
leads to autophagy upregulation in vitro. This disruption can be
mediated by multi-site phosphorylation of the non-structured loop
of Bcl-2,.sup.26 regulatory phosphorylation of the BH3 domain (an
amphipathic alpha helix) of Beclin 1 that reduces its affinity for
Bcl-2,.sup.29-30 or BH3-only proteins that competitively disrupt
Beclin 1/Bcl-2 binding..sup.31 Genetically engineered mice with
mutations in the Bcl-2 phosphorylation sites required for
disruption of Beclin 1/Bcl-2 binding are deficient in starvation-
and exercise-induced autophagy in vivo, and fail to experience
beneficial metabolic effects of long-term exercise
training..sup.32
Conversely, knock-in mice with a point mutation in Beclin 1 (F123A
inhuman protein, F121A in mouse protein) that reduces its binding
affinity in vitro for Bcl-2 and Bcl-xL results in increased
constitutive autophagy in multiple tissues, including brain, heart,
muscle, liver, mammary gland and kidney..sup.17-19 These mice
demonstrate increased longevity and diminished aging-related
phenotypes, particularly age-related renal and cardiac pathological
changes and age-related spontaneous tumorigenesis. The Beclin 1
knock-in mutation also decreases the accumulation of amyloid
oligomers and improves cognitive function and survival in a mouse
model of Alzheimers-like disease.sup.18 and decreases the incidence
of breast cancer in a mouse model of HER2-driven
tumorigenesis..sup.19
Thus, there is compelling genetic evidence that Beclin 1/Bcl-2
interaction serves as an important checkpoint for autophagy
induction in vivo. Importantly, the long-term disruption of this
complex is not only safe in mice, but it also improves healthspan,
extends lifespan, and protects against neurodegenerative diseases
and cancers. These in vivo findings, taken together with the
extensive in vitro data about the role of Beclin 1/Bcl-2 in
autophagy regulation,.sup.25, 30 provide a strong rationale for the
development of new autophagy-inducing strategies that target Beclin
1/Bcl-2 binding.
An important challenge of this approach is posed by the overlapping
modality by which the BH3 domain of the autophagic protein Beclin 1
and that of pro-apoptotic proteins bind to the conserved
hydrophobic grooves of Bcl-2/Bcl-xL..sup.33-34 BH3 mimetics (e.g.
ABT-737, ABT-263, ABT-199) have been developed that disrupt Bcl-2
(and/or Bcl-xL) binding of Beclin 1 (and thereby induce
autophagy).sup.31, 35 but they also disrupt binding between
anti-apoptotic Bcl-2 family members and pro-apoptotic BH3 domains
(and thereby induce apoptosis)..sup.36 These compounds were
optimized for their apoptosis-inducing activity and use as
potential cancer chemotherapeutic agents. However, from the
standpoint of treating infectious diseases and neurodegeneration
and preventing aging (as well as other pathophysiological contexts
where upregulation of autophagy may be beneficial), it would be
desirable to develop agents that selectively disrupt Beclin 1/Bcl-2
binding but not Bcl-2 family member/pro-apoptotic family member
binding to selectively induce autophagy (a pro-survival pathway)
without inducing apoptosis. This may be technically feasible since
the binding affinity of the BH3 domain of Beclin 1 to Bcl-xL is
considerably lower than that of pro-apoptotic BH3 family
members,.sup.34, 37-38 thus providing a potential therapeutic
window for selective disruption of Beclin 1 binding to
Bcl-2/Bcl-xL. Furthermore, detailed biochemical and biophysical
analyses of BH3 peptide binding to Bcl-2 family members indicate
that differences in the precise binding modalities of different BH3
domains exist that might be exploited therapeutically..sup.33-34,
39 Therefore, we sought to identify novel autophagy-inducing drugs
that target the Beclin 1/Bcl-2 interaction without perturbing the
binding of Bcl-2 to pro-apoptotic BH3 domain-containing
molecules.
We employed a high-throughput screening (HTS) platform using either
a cell-based split-luciferase or in vitro AlphaLISA assays to
identify novel Beclin 1/Bcl-2 binding inhibitors. Using chemical
libraries comprising .about.300,000 compounds from the UT
Southwestern Medical Center and the Broad Institute of MIT and
Harvard, we identified three small molecule compounds that both
disrupt Beclin 1/Bcl-2 interaction in the cell-based
split-luciferase assay and directly inhibit Beclin 1/Bcl-2
interaction in the in vitro AlphaLISA assay with micromolar IC50
values. These three compounds induce autophagic flux in cells at
concentrations that do not decrease cell viability. Our biochemical
data indicate that these compounds inhibit the interaction between
the BH3 domain of Beclin 1 and Bcl-2 without affecting the
interaction between the BH3 domain of the pro-apoptotic protein,
Bax, and Bcl-2 or between the BH3-only protein, Bim, and Bcl-2,
indicating that these compounds are selective Beclin 1/Bcl-2
inhibitors. Overall, this screening program identified compounds
that selectively disrupt Beclin 1/Bcl-2 interaction.
SUMMARY OF THE INVENTION
Autophagy plays a crucial role in cellular homeostasis,
development, immunity, tumor suppression, metabolism, prevention of
neurodegeneration, and lifespan extension. Thus, pharmacological
stimulation of autophagy provides effective approach for preventing
or treating certain human diseases and/or aging. Using
split-luciferase and AlphaLISA assays and SAR development we
developed small molecules that selectively inhibit the Beclin
1/Bcl-2 protein-protein interaction (versus inhibiting other
Bcl-2/BH3-domain containing protein-protein interactions that would
induce apoptosis). These small molecule disruptors of
Beclin-1/Bcl-2 protein-protein interactions induce autophagy and
hence are useful for treating a variety of indications where
stimulation (induction) of autophagy is therapeutically useful,
including cancer, infection (including Zika) immunity,
neurodegeneration, longevity.
In aspects the invention provides: A method of selectively inducing
autophagy, comprising treating a person in need thereof with a
pharmaceutical composition comprising a compound of structure I; A
method of selectively inhibiting binding of Beclin 1 BH3 domain to
anti-apoptotic Bcl-2 BH3 domain, but not binding of pro-apoptotic
Bax BH3 domain or pro-apoptotic Bim BH3 protein to Bcl-2 BH3
domain, comprising treating a person in need thereof with a
pharmaceutical composition comprising a compound of structure I; A
pharmaceutical composition formulated for administration to a
person in need thereof, comprising a compound of structure I; and A
compound, excluding library compound SW076956, of structure I:
##STR00001## wherein
R1 is optionally substituted C5 or C6 aryl or heteroaryl;
R1 is optionally substituted C5 or C6 aryl or heteroaryl, having
1-3 heteroatoms selected from N, O and S;
R1 is optionally substituted pyrrole, furan, thiophene, imidazole,
pyrazole, oxazole, isoxazole, thiazole, isothiazole, triazole,
furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, pyridine,
pyran, thiopyran, oxazine, thiazine, dioxine, dithiin and triazone;
or
R1 is optionally substituted 2- or 3-furanyl or phenyl, 2, 3 or
4-pyridine;
R2 is 0-5 substituents independently selected from optionally
substituted heteroatom or optionally substituted, optionally
hetero-, optionally cyclic C1-C10 hydrocarbyl;
R2 is 0-5 substituents independently selected from halogen, amide,
amine, imine, imide, nitro, nitroso, nitrate, nitrite, cyano,
hydroxyl or hydrocarbyloxy, or aldehyde, ketone, carboxyl, ether,
ester, alkyl, alkenyl, alkynyl, sulfinyl, sulfonyl;
R2 is 0-5 substituents selected from halogen, NO.sub.2, N, C1-C4
alkyl (Me, Et, cPr, iPr, cBu, tBu), C1-C4 alkoxy, C1-C4 carbonyl,
C1-C4 carboxyl, C1-C4 cyano, C1-C4 sulfinyl, C1-C4 sulfonyl, each
optionally fluorinated; or
R2 is 1 para substituent selected from halogen, NO.sub.2, N, C1-C4
alkyl, C1-C4 alkoxy, C1-C4 carbonyl, C1-C4 carboxyl, C1-C4 cyano,
C1-C4 sulfinyl, C1-C4 sulfonyl, each optionally fluorinated;
R3 is 0-4 substituents independently selected from optionally
substituted heteroatom or optionally substituted, optionally
hetero-, optionally cyclic C1-C10 hydrocarbyl;
R3 is 0-4 substituents independently selected from halogen, amide,
amine, imine, imide, nitro, nitroso, nitrate, nitrite, cyano,
hydroxyl or hydrocarbyloxy, or aldehyde, ketone, carboxyl, ether,
ester, alkyl, alkenyl, alkynyl, sulfinyl, sulfonyl;
R3 is 0-4 substituents selected from halogen, NO.sub.2, N, C1-C4
alkyl (Me, Et, cPr, iPr, cBu, tBu), C1-C4 alkoxy, C1-C4 carbonyl,
C1-C4 carboxyl, C1-C4 cyano, C1-C4 sulfinyl, C1-C4 sulfonyl, each
optionally fluorinated; or
R3 is 0 substituents;
R4 is H, optionally substituted heteroatom or optionally
substituted, optionally hetero-, optionally cyclic C1-C10
hydrocarbyl, or
R4 is H, hydroxyl or hydrocarbyloxy, or aldehyde, ketone, carboxyl,
ether, ester, alkyl, alkenyl, alkynyl, sulfinyl, sulfonyl;
R4 is H or C1-C4 alkyl (Me, Et, cPr, iPr, cBu, tBu), C1-C4 alkoxy,
C1-C4 carbonyl, C1-C4 carboxyl, C1-C4 cyano, C1-C4 sulfinyl, C1-C4
sulfonyl, each optionally fluorinated; or
R4 is H;
R5 is optionally substituted heteroatom or optionally substituted,
optionally hetero-, optionally cyclic C1-C10 hydrocarbyl;
R5 is hydroxyl or hydrocarbyloxy, or aldehyde, ketone, carboxyl,
ether, ester, alkyl, alkenyl, alkynyl, sulfinyl, sulfonyl;
R5 is C1-C4 alkyl (Me, Et, cPr, iPr, cBu, tBu), C1-C4 alkoxy, C1-C4
carbonyl, C1-C4 carboxyl, C1-C4 cyano, C1-C4 sulfinyl, C1-C4
sulfonyl, each optionally fluorinated; or
R5 is sulfonyl or ester, SO.sub.2R or CO.sub.2R, wherein R is C1-C4
alkyl, optionally fluorinated;
wherein R4 and R5 may be joined in a heterocylic ring;
or a salt, hydrate, or stereoisomer thereof.
The invention includes all combinations of recited particular
embodiments as if each combination had been laboriously
recited.
DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
The term "alkyl" refers to a hydrocarbon group selected from linear
and branched saturated hydrocarbon groups of 1-18, or 1-12, or 1-6
carbon atoms. Examples of the alkyl group include methyl, ethyl,
1-propyl or n-propyl ("n-Pr"), 2-propyl or isopropyl ("i-Pr"),
1-butyl or n-butyl ("n-Bu"), 2-methyl-1-propyl or isobutyl
("i-Bu"), 1-methylpropyl or s-butyl ("s-Bu"), and 1,1-dimethylethyl
or t-butyl ("t-Bu"). Other examples of the alkyl group include
1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,
3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl,
2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,
3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl and
3,3-dimethyl-2-butyl groups.
Lower alkyl means 1-8, preferably 1-6, more preferably 1-4 carbon
atoms; lower alkenyl or alkynyl means 2-8, 2-6 or 2-4 carbon
atoms.
The term "alkenyl" refers to a hydrocarbon group selected from
linear and branched hydrocarbon groups comprising at least one
C.dbd.C double bond and of 2-18, or 2-12, or 2-6 carbon atoms.
Examples of the alkenyl group may be selected from ethenyl or
vinyl, prop-1-enyl, prop-2-enyl, 2-methylprop-1-enyl, but-1-enyl,
but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-diene,
hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, and hexa-1,3-dienyl
groups.
The term "alkynyl" refers to a hydrocarbon group selected from
linear and branched hydrocarbon group, comprising at least one
C.dbd.C triple bond and of 2-18, or 2-12, or 2-6 carbon atoms.
Examples of the alkynyl group include ethynyl, 1-propynyl,
2-propynyl (propargyl), 1-butynyl, 2-butynyl, and 3-butynyl
groups.
The term "cycloalkyl" refers to a hydrocarbon group selected from
saturated and partially unsaturated cyclic hydrocarbon groups,
comprising monocyclic and polycyclic (e.g., bicyclic and tricyclic)
groups. For example, the cycloalkyl group may be of 3-12, or 3-8,
or 3-6 carbon atoms. Even further for example, the cycloalkyl group
may be a monocyclic group of 3-12, or 3-8, or 3-6 carbon atoms.
Examples of the monocyclic cycloalkyl group include cyclopropyl,
cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,
1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl,
cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl
groups. Examples of the bicyclic cycloalkyl groups include those
having 7-12 ring atoms arranged as a bicycle ring selected from
[4,4], [4,5], [5,5], [5,6] and [6,6] ring systems, or as a bridged
bicyclic ring selected from bicyclo[2.2.1]heptane,
bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. The ring may be
saturated or have at least one double bond (i.e. partially
unsaturated), but is not fully conjugated, and is not aromatic, as
aromatic is defined herein.
The term "aryl" herein refers to a group selected from: 5- and
6-membered carbocyclic aromatic rings, for example, phenyl;
bicyclic ring systems such as 7-12 membered bicyclic ring systems
wherein at least one ring is carbocyclic and aromatic, selected,
for example, from naphthalene, indane, and
1,2,3,4-tetrahydroquinoline; and tricyclic ring systems such as
10-15 membered tricyclic ring systems wherein at least one ring is
carbocyclic and aromatic, for example, fluorene.
For example, the aryl group is selected from 5- and 6-membered
carbocyclic aromatic rings fused to a 5- to 7-membered cycloalkyl
or heterocyclic ring optionally comprising at least one heteroatom
selected from N, O, and S, provided that the point of attachment is
at the carbocyclic aromatic ring when the carbocyclic aromatic ring
is fused with a heterocyclic ring, and the point of attachment can
be at the carbocyclic aromatic ring or at the cycloalkyl group when
the carbocyclic aromatic ring is fused with a cycloalkyl group.
Bivalent radicals formed from substituted benzene derivatives and
having the free valences at ring atoms are named as substituted
phenylene radicals. Bivalent radicals derived from univalent
polycyclic hydrocarbon radicals whose names end in "-yl" by removal
of one hydrogen atom from the carbon atom with the free valence are
named by adding "-idene" to the name of the corresponding univalent
radical, e.g., a naphthyl group with two points of attachment is
termed naphthylidene. Aryl, however, does not encompass or overlap
with heteroaryl, separately defined below. Hence, if one or more
carbocyclic aromatic rings are fused with a heterocyclic aromatic
ring, the resulting ring system is heteroaryl, not aryl, as defined
herein.
The term "halogen" or "halo" refers to F, Cl, Br or I.
The term "heteroalkyl" refers to alkyl comprising at least one
heteroatom.
The term "heteroaryl" refers to a group selected from:
5- to 7-membered aromatic, monocyclic rings comprising 1, 2, 3 or 4
heteroatoms selected from N, O, and S, with the remaining ring
atoms being carbon;
8- to 12-membered bicyclic rings comprising 1, 2, 3 or 4
heteroatoms, selected from N, O, and S, with the remaining ring
atoms being carbon and wherein at least one ring is aromatic and at
least one heteroatom is present in the aromatic ring; and
11- to 14-membered tricyclic rings comprising 1, 2, 3 or 4
heteroatoms, selected from N, O, and S, with the remaining ring
atoms being carbon and wherein at least one ring is aromatic and at
least one heteroatom is present in an aromatic ring.
For example, the heteroaryl group includes a 5- to 7-membered
heterocyclic aromatic ring fused to a 5- to 7-membered cycloalkyl
ring. For such fused, bicyclic heteroaryl ring systems wherein only
one of the rings comprises at least one heteroatom, the point of
attachment may be at the heteroaromatic ring or at the cycloalkyl
ring.
When the total number of S and O atoms in the heteroaryl group
exceeds 1, those heteroatoms are not adjacent to one another. In
some embodiments, the total number of S and O atoms in the
heteroaryl group is not more than 2. In some embodiments, the total
number of S and O atoms in the aromatic heterocycle is not more
than 1.
Examples of the heteroaryl group include, but are not limited to,
(as numbered from the linkage position assigned priority 1) pyridyl
(such as 2-pyridyl, 3-pyridyl, or 4-pyridyl), cinnolinyl,
pyrazinyl, 2,4-pyrimidinyl, 3,5-pyrimidinyl, 2,4-imidazolyl,
imidazopyridinyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl,
thiadiazolyl, tetrazolyl, thienyl, triazinyl, benzothienyl, furyl,
benzofuryl, benzoimidazolyl, indolyl, isoindolyl, indolinyl,
phthalazinyl, pyrazinyl, pyridazinyl, pyrrolyl, triazolyl,
quinolinyl, isoquinolinyl, pyrazolyl, pyrrolopyridinyl (such as
1H-pyrrolo[2,3-b]pyridin-5-yl), pyrazolopyridinyl (such as
1H-pyrazolo[3,4-b]pyridin-5-yl), benzoxazolyl (such as
benzo[d]oxazol-6-yl), pteridinyl, purinyl, 1-oxa-2,3-diazolyl,
1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa-3,4-diazolyl,
1-thia-2,3-diazolyl, 1-thia-2,4-diazolyl, 1-thia-2,5-diazolyl,
1-thia-3,4-diazolyl, furazanyl, benzofurazanyl, benzothiophenyl,
benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,
naphthyridinyl, furopyridinyl, benzothiazolyl (such as
benzo[d]thiazol-6-yl), indazolyl (such as 1H-indazol-5-yl) and
5,6,7,8-tetrahydroisoquinoline.
The term "heterocyclic" or "heterocycle" or "heterocyclyl" refers
to a ring selected from 4- to 12-membered monocyclic, bicyclic and
tricyclic, saturated and partially unsaturated rings comprising at
least one carbon atoms in addition to 1, 2, 3 or 4 heteroatoms,
selected from oxygen, sulfur, and nitrogen. "Heterocycle" also
refers to a 5- to 7-membered heterocyclic ring comprising at least
one heteroatom selected from N, O, and S fused with 5-, 6-, and/or
7-membered cycloalkyl, carbocyclic aromatic or heteroaromatic ring,
provided that the point of attachment is at the heterocyclic ring
when the heterocyclic ring is fused with a carbocyclic aromatic or
a heteroaromatic ring, and that the point of attachment can be at
the cycloalkyl or heterocyclic ring when the heterocyclic ring is
fused with cycloalkyl.
"Heterocycle" also refers to an aliphatic spirocyclic ring
comprising at least one heteroatom selected from N, O, and S,
provided that the point of attachment is at the heterocyclic ring.
The rings may be saturated or have at least one double bond (i.e.
partially unsaturated). The heterocycle may be substituted with
oxo. The point of the attachment may be carbon or heteroatom in the
heterocyclic ring. A heterocyle is not a heteroaryl as defined
herein.
Examples of the heterocycle include, but not limited to, (as
numbered from the linkage position assigned priority 1)
1-pyrrolidinyl, 2-pyrrolidinyl, 2,4-imidazolidinyl,
2,3-pyrazolidinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl,
4-piperidinyl, 2,5-piperazinyl, pyranyl, 2-morpholinyl,
3-morpholinyl, oxiranyl, aziridinyl, thiiranyl, azetidinyl,
oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl,
dihydropyridinyl, tetrahydropyridinyl, thiomorpholinyl, thioxanyl,
piperazinyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl,
thiepanyl, 1,4-oxathianyl, 1,4-dioxepanyl, 1,4-oxathiepanyl,
1,4-oxaazepanyl, 1,4-dithiepanyl, 1,4-thiazepanyl and 1,4-diazepane
1,4-dithianyl, 1,4-azathianyl, oxazepinyl, diazepinyl, thiazepinyl,
dihydrothienyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl,
tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl,
1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,
4H-pyranyl, 1,4-dioxanyl, 1,3-dioxolanyl, pyrazolinyl,
pyrazolidinyl, dithianyl, dithiolanyl, imidazolinyl, pyrimidinonyl,
1,1-dioxo-thiomorpholinyl, 3-azabicyco[3.1.0]hexanyl,
3-azabicyclo[4.1.0]heptanyl and azabicyclo[2.2.2]hexanyl.
Substituted heterocycle also includes ring systems substituted with
one or more oxo moieties, such as piperidinyl N-oxide,
morpholinyl-N-oxide, 1-oxo-1-thiomorpholinyl and
1,1-dioxo-1-thiomorpholinyl.
Substituents are selected from: halogen, --R', --OR', .dbd.O,
.dbd.NR', .dbd.N--OR', --NR'R'', --SR', --SiR'R''R''', --OC(O)R',
--C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR'--C(O)NR''R''', --NR'--SO.sub.2NR''', --NR''CO.sub.2R',
--NH--C(NH.sub.2).dbd.NH, --NR'C(NH.sub.2).dbd.NH,
--NH--C(NH.sub.2).dbd.NR', --S(O)R', --SO.sub.2R',
--SO.sub.2NR'R'', --NR''SO.sub.2R, --CN and --NO.sub.2, --N.sub.3,
--CH(Ph).sub.2, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl,
in a number ranging from zero to three, with those groups having
zero, one or two substituents being particularly preferred. R', R''
and R''' each independently refer to hydrogen, unsubstituted
(C1-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted
with one to three halogens, unsubstituted alkyl, alkoxy or
thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When R' and R'' are
attached to the same nitrogen atom, they can be combined with the
nitrogen atom to form a 5-, 6- or 7-membered ring. Hence, --NR'R''
includes 1-pyrrolidinyl and 4-morpholinyl, "alkyl" includes groups
such as trihaloalkyl (e.g., --CF.sub.3 and --CH.sub.2CF.sub.3), and
when the aryl group is 1,2,3,4-tetrahydronaphthalene, it may be
substituted with a substituted or unsubstituted
(C3-C7)spirocycloalkyl group. The (C3-C7)spirocycloalkyl group may
be substituted in the same manner as defined herein for
"cycloalkyl".
Preferred substituents are selected from: halogen, --R', --OR',
.dbd.O, --NR'R'', --SR', --SiR'R''R''', --OC(O)R', --C(O)R',
--CO.sub.2R', --CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR''CO.sub.2R', --NR'--SO.sub.2NR''R''', --S(O)R', --SO.sub.2R',
--SO.sub.2NR'R'', --NR''SO.sub.2R, --CN and --NO.sub.2,
perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, where R' and R''
are as defined above.
The term "fused ring" herein refers to a polycyclic ring system,
e.g., a bicyclic or tricyclic ring system, in which two rings share
only two ring atoms and one bond in common. Examples of fused rings
may comprise a fused bicyclic cycloalkyl ring such as those having
from 7 to 12 ring atoms arranged as a bicyclic ring selected from
[4,4], [4,5], [5,5], [5,6] and [6,6] ring systems as mentioned
above; a fused bicyclic aryl ring such as 7 to 12 membered bicyclic
aryl ring systems as mentioned above, a fused tricyclic aryl ring
such as 10 to 15 membered tricyclic aryl ring systems mentioned
above; a fused bicyclic heteroaryl ring such as 8- to 12-membered
bicyclic heteroaryl rings as mentioned above, a fused tricyclic
heteroaryl ring such as 11- to 14-membered tricyclic heteroaryl
rings as mentioned above; and a fused bicyclic or tricyclic
heterocyclyl ring as mentioned above.
The compounds may contain an asymmetric center and may thus exist
as enantiomers. Where the compounds possess two or more asymmetric
centers, they may additionally exist as diastereomers. Enantiomers
and diastereomers fall within the broader class of stereoisomers.
All such possible stereoisomers as substantially pure resolved
enantiomers, racemic mixtures thereof, as well as mixtures of
diastereomers are intended to be included. All stereoisomers of the
compounds and/or pharmaceutically acceptable salts thereof are
intended to be included. Unless specifically mentioned otherwise,
reference to one isomer applies to any of the possible isomers.
Whenever the isomeric composition is unspecified, all possible
isomers are included.
The compounds of the invention may also contain unnatural
proportions of atomic isotopes at one or more of the atoms that
constitute such compounds, such as deuterium, e.g. --CD.sub.3,
CD.sub.2H or CDH.sub.2 in place of methyl. For example, the
compounds may be radiolabeled with radioactive isotopes, such as
for example tritium (.sup.3H), iodine-125 (.sup.125I) or carbon-14
(.sup.14C). All isotopic variations of the compounds of the
invention, whether radioactive or not, are intended to be
encompassed within the scope of the invention.
The term "substantially pure" means that the target stereoisomer
contains no more than 35%, such as no more than 30%, further such
as no more than 25%, even further such as no more than 20%, by
weight of any other stereoisomer(s). In some embodiments, the term
"substantially pure" means that the target stereoisomer contains no
more than 10%, for example, no more than 5%, such as no more than
1%, by weight of any other stereoisomer(s).
When compounds contain olefin double bonds, unless specified
otherwise, such double bonds are meant to include both E and Z
geometric isomers.
Some of the compounds may exist with different points of attachment
of hydrogen, referred to as tautomers. For example, compounds
including carbonyl --CH.sub.2C(O)-- groups (keto forms) may undergo
tautomerism to form hydroxyl --CH.dbd.C(OH)-- groups (enol forms).
Both keto and enol forms, individually as well as mixtures thereof,
are also intended to be included where applicable.
It may be advantageous to separate reaction products from one
another and/or from starting materials. The desired products of
each step or series of steps is separated and/or purified
(hereinafter separated) to the desired degree of homogeneity by the
techniques common in the art. Typically such separations involve
multiphase extraction, crystallization from a solvent or solvent
mixture, distillation, sublimation, or chromatography.
Chromatography can involve any number of methods including, for
example: reverse-phase and normal phase; size exclusion; ion
exchange; high, medium and low pressure liquid chromatography
methods and apparatus; small scale analytical; simulated moving bed
("SMB") and preparative thin or thick layer chromatography, as well
as techniques of small scale thin layer and flash chromatography.
One skilled in the art will apply techniques most likely to achieve
the desired separation.
Diastereomeric mixtures can be separated into their individual
diastereomers on the basis of their physical chemical differences
by methods well known to those skilled in the art, such as by
chromatography and/or fractional crystallization. Enantiomers can
be separated by converting the enantiomeric mixture into a
diastereomeric mixture by reaction with an appropriate optically
active compound (e.g., chiral auxiliary such as a chiral alcohol or
Mosher's acid chloride), separating the diastereomers and
converting (e.g., hydrolyzing) the individual diastereoisomers to
the corresponding pure enantiomers. Enantiomers can also be
separated by use of a chiral HPLC column.
A single stereoisomer, e.g., a substantially pure enantiomer, may
be obtained by resolution of the racemic mixture using a method
such as formation of diastereomers using optically active resolving
agents (Eliel, E. and Wilen, S. Stereochemistry of Organic
Compounds. New York: John Wiley & Sons, Inc., 1994; Lochmuller,
C. H., et al. "Chromatographic resolution of enantiomers: Selective
review." J. Chromatogr., 113(3) (1975): pp. 283-302). Racemic
mixtures of chiral compounds of the invention can be separated and
isolated by any suitable method, including: (1) formation of ionic,
diastereomeric salts with chiral compounds and separation by
fractional crystallization or other methods, (2) formation of
diastereomeric compounds with chiral derivatizing reagents,
separation of the diastereomers, and conversion to the pure
stereoisomers, and (3) separation of the substantially pure or
enriched stereoisomers directly under chiral conditions. See:
Wainer, Irving W., Ed. Drug Stereochemistry: Analytical Methods and
Pharmacology. New York: Marcel Dekker, Inc., 1993.
"Pharmaceutically acceptable salts" include, but are not limited to
salts with inorganic acids, selected, for example, from
hydrochlorates, phosphates, diphosphates, hydrobromates, sulfates,
sulfinates, and nitrates; as well as salts with organic acids,
selected, for example, from malates, maleates, fumarates,
tartrates, succinates, citrates, lactates, methanesulfonates,
p-toluenesulfonates, 2-hydroxyethylsulfonates, benzoates,
salicylates, stearates, alkanoates such as acetate, and salts with
HOOC--(CH.sub.2)n-COOH, wherein n is selected from 0 to 4.
Similarly, examples of pharmaceutically acceptable cations include,
but are not limited to, sodium, potassium, calcium, aluminum,
lithium, and ammonium.
In addition, if a compound is obtained as an acid addition salt,
the free base can be obtained by basifying a solution of the acid
salt. Conversely, if the product is a free base, an addition salt,
such as a pharmaceutically acceptable addition salt, may be
produced by dissolving the free base in a suitable organic solvent
and treating the solution with an acid, in accordance with
conventional procedures for preparing acid addition salts from base
compounds. Those skilled in the art will recognize various
synthetic methodologies that may be used without undue
experimentation to prepare non-toxic pharmaceutically acceptable
addition salts.
"Treating," "treat," or "treatment" refers to administering at
least one compound and/or at least one stereoisomer thereof, and/or
at least one pharmaceutically acceptable salt thereof to a subject
in recognized need thereof.
An "effective amount" refers to an amount of at least one compound
and/or at least one stereoisomer thereof, and/or at least one
pharmaceutically acceptable salt thereof effective to "treat" a
disease or disorder in a subject, and that will elicit, to some
significant extent, the biological or medical response of a tissue,
system, animal or human that is being sought, such as when
administered, is sufficient to prevent development of, or alleviate
to some extent, one or more of the symptoms of the condition or
disorder being treated. The therapeutically effective amount will
vary depending on the compound, the disease and its severity and
the age, weight, etc., of the mammal to be treated.
The term "at least one substituent" includes, for example, from 1
to 4, such as from 1 to 3, further as 1 or 2, substituents. For
example, "at least one substituent R" herein includes from 1 to 4,
such as from 1 to 3, further as 1 or 2, substituents selected from
the list of R as described herein.
The subject compounds and stereoisomers thereof, and
pharmaceutically acceptable salts thereof may be employed alone or
in combination with at least one other therapeutic agent for
treatment. In some embodiments, the compounds, stereoisomers
thereof, and pharmaceutically acceptable salts thereof can be used
in combination with at least one additional therapeutic agent. The
compound and/or one pharmaceutically acceptable salt disclosed
herein may be administered with the at least one other therapeutic
agent in a single dosage form or as a separate dosage form. When
administered as a separate dosage form, the at least one other
therapeutic agent may be administered prior to, at the same time
as, or following administration of the compound and/or one
pharmaceutically acceptable salt disclosed herein.
Also provided is a composition comprising a subject compound and
stereoisomers thereof, and pharmaceutically acceptable salts
thereof, and at least one pharmaceutically acceptable carrier.
The composition comprising a subject compound and stereoisomers
thereof, and pharmaceutically acceptable salts thereof can be
administered in various known manners, such as orally, topically,
rectally, parenterally, by inhalation spray, or via an implanted
reservoir, although the most suitable route in any given case will
depend on the particular host, and nature and severity of the
conditions for which the active ingredient is being administered.
The term "parenteral" as used herein includes subcutaneous,
intracutaneous, intravenous, intramuscular, intraarticular,
intraarterial, intrasynovial, intrasternal, intrathecal,
intralesional and intracranial injection or infusion techniques.
The compositions disclosed herein may be conveniently presented in
unit dosage form and prepared by any of the methods well known in
the art.
The subject compounds and stereoisomers thereof, and
pharmaceutically acceptable salts thereof can be administered
orally in solid dosage forms, such as capsules, tablets, troches,
dragees, granules and powders, or in liquid dosage forms, such as
elixirs, syrups, emulsions, dispersions, and suspensions. The
subject compounds and stereoisomers thereof, and pharmaceutically
acceptable salts thereof disclosed herein can also be administered
parenterally, in sterile liquid dosage forms, such as dispersions,
suspensions or solutions. Other dosages forms that can also be used
to administer the subject compounds and stereoisomers thereof, and
pharmaceutically acceptable salts thereof disclosed herein as an
ointment, cream, drops, transdermal patch or powder for topical
administration, as an ophthalmic solution or suspension formation,
i.e., eye drops, for ocular administration, as an aerosol spray or
powder composition for inhalation or intranasal administration, or
as a cream, ointment, spray or suppository for rectal or vaginal
administration.
Gelatin capsules containing the compound and/or the at least one
pharmaceutically acceptable salt thereof disclosed herein and
powdered carriers, such as lactose, starch, cellulose derivatives,
magnesium stearate, stearic acid, and the like, can also be used.
Similar diluents can be used to make compressed tablets. Both
tablets and capsules can be manufactured as sustained release
products to provide for continuous release of medication over a
period of time. Compressed tablets can be sugar coated or film
coated to mask any unpleasant taste and protect the tablet from the
atmosphere, or enteric coated for selective disintegration in the
gastrointestinal tract.
Liquid dosage forms for oral administration can further comprise at
least one agent selected from coloring and flavoring agents to
increase patient acceptance.
In general, water, a suitable oil, saline, aqueous dextrose
(glucose), and related sugar solutions and glycols such as
propylene glycol or polyethylene gycols can be examples of suitable
carriers for parenteral solutions. Solutions for parenteral
administration may comprise a water soluble salt of the at least
one compound describe herein, at least one suitable stabilizing
agent, and if necessary, at least one buffer substance.
Antioxidizing agents such as sodium bisulfite, sodium sulfite, or
ascorbic acid, either alone or combined, can be examples of
suitable stabilizing agents. Citric acid and its salts and sodium
EDTA can also be used as examples of suitable stabilizing agents.
In addition, parenteral solutions can further comprise at least one
preservative, selected, for example, from benzalkonium chloride,
methyl- and propylparaben, and chlorobutanol.
A pharmaceutically acceptable carrier is, for example, selected
from carriers that are compatible with active ingredients of the
composition (and in some embodiments, capable of stabilizing the
active ingredients) and not deleterious to the subject to be
treated. For example, solubilizing agents, such as cyclodextrins
(which can form specific, more soluble complexes with the at least
one compound and/or at least one pharmaceutically acceptable salt
disclosed herein), can be utilized as pharmaceutical excipients for
delivery of the active ingredients. Examples of other carriers
include colloidal silicon dioxide, magnesium stearate, cellulose,
sodium lauryl sulfate, and pigments such as D&C Yellow #10.
Suitable pharmaceutically acceptable carriers are described in
Remington's Pharmaceutical Sciences, A. Osol, and other reference
texts in the art.
For administration by inhalation, the subject compounds and
stereoisomers thereof, and pharmaceutically acceptable salts
thereof may be conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or nebulisers. The
subject compounds and stereoisomers thereof, and pharmaceutically
acceptable salts thereof may also be delivered as powders, which
may be formulated and the powder composition may be inhaled with
the aid of an insufflation powder inhaler device. One exemplary
delivery system for inhalation can be metered dose inhalation (MDI)
aerosol, which may be formulated as a suspension or solution of a
subject compound and stereoisomers thereof, and pharmaceutically
acceptable salts thereof disclosed herein in at least one suitable
propellant, selected, for example, from fluorocarbons and
hydrocarbons.
For ocular administration, an ophthalmic preparation may be
formulated with an appropriate weight percentage of a solution or
suspension of the subject compound and stereoisomers thereof, and
pharmaceutically acceptable salts thereof in an appropriate
ophthalmic vehicle, such that the subject compound and
stereoisomers thereof, and at least one pharmaceutically acceptable
salts thereof is maintained in contact with the ocular surface for
a sufficient time period to allow the compound to penetrate the
corneal and internal regions of the eye.
Useful pharmaceutical dosage-forms for administration of the
subject compounds and stereoisomers thereof, and pharmaceutically
acceptable salts thereof disclosed herein include, but are not
limited to, hard and soft gelatin capsules, tablets, parenteral
injectables, and oral suspensions.
The dosage administered will be dependent on factors, such as the
age, health and weight of the recipient, the extent of disease,
type of concurrent treatment, if any, frequency of treatment, and
the nature of the effect desired. In general, a daily dosage of the
active ingredient can vary, for example, from 0.1 to 2000
milligrams per day. For example, 10-500 milligrams once or multiple
times per day may be effective to obtain the desired results.
In some embodiments, a large number of unit capsules can be
prepared by filling standard two-piece hard gelatin capsules each
with, for example, 100 milligrams of the subject compound and
stereoisomers thereof, and pharmaceutically acceptable salt thereof
disclosed herein in powder, 150 milligrams of lactose, 50
milligrams of cellulose, and 6 milligrams magnesium stearate.
In some embodiments, a mixture of the compound, stereoisomers
thereof, and pharmaceutically acceptable salts thereof a digestible
oil such as soybean oil, cottonseed oil or olive oil can be
prepared and injected by means of a positive displacement pump into
gelatin to form soft gelatin capsules containing 100 milligrams of
the active ingredient. The capsules are washed and dried.
In some embodiments, a large number of tablets can be prepared by
conventional procedures so that the dosage unit comprises, for
example, 100 milligrams of the compound, stereoisomers thereof, and
pharmaceutically acceptable salts thereof, 0.2 milligrams of
colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275
milligrams of microcrystalline cellulose, 11 milligrams of starch
and 98.8 milligrams of lactose. Appropriate coatings may be applied
to increase palatability or delay absorption.
In some embodiments, a parenteral composition suitable for
administration by injection can be prepared by stirring 1.5% by
weight of the compound and/or at least an enantiomer, a
diastereomer, or pharmaceutically acceptable salt thereof disclosed
herein in 10% by volume propylene glycol. The solution is made to
the expected volume with water for injection and sterilized.
In some embodiment, an aqueous suspension can be prepared for oral
administration. For example, each 5 milliliters of an aqueous
suspension comprising 100 milligrams of finely divided compound,
stereoisomers thereof, and pharmaceutically acceptable salts
thereof, 100 milligrams of sodium carboxymethyl cellulose, 5
milligrams of sodium benzoate, 1.0 grams of sorbitol solution,
U.S.P., and 0.025 milliliters of vanillin can be used.
The same dosage forms can generally be used when the compound,
stereoisomers thereof, and pharmaceutically acceptable salts
thereof are administered stepwise or in conjunction with at least
one other therapeutic agent. When drugs are administered in
physical combination, the dosage form and administration route
should be selected depending on the compatibility of the combined
drugs. Thus the term coadministration is understood to include the
administration of at least two agents concomitantly or
sequentially, or alternatively as a fixed dose combination of the
at least two active components.
The compounds, stereoisomers thereof, and pharmaceutically
acceptable salt thereof disclosed herein can be administered as the
sole active ingredient or in combination with at least one second
active ingredient.
The subject compounds are incorporated into pharmaceutical
compositions or formulations. The compositions will contain
pharmaceutically acceptable diluents and/or carriers, i. e.
diluents or carriers that are physiologically compatible and
substantially free from pathogenic impurities. Suitable excipients
or carriers and methods for preparing administrable compositions
are known or apparent to those skilled in the art and are described
in more detail in such publications as Remington's Pharmaceutical
Science, Mack Publishing Co, NJ (1991). The compositions may also
be in the form of controlled release or sustained release
compositions as known in the art. For many applications the subject
compounds are administered for morning/daytime dosing, with off
period at night.
The subject compounds may be used per se, or in the form of their
pharmaceutically acceptable salts, such as hydrochlorides,
hydrobromides, acetates, sulfates, citrates, carbonates,
trifluoroacetates and the like. When compounds contain relatively
acidic functionalities, salts can be obtained by addition of the
desired base, either neat or in a suitable inert solvent.
Examples of pharmaceutically acceptable base addition salts include
sodium, potassium, calcium, ammonium, organic amino, or magnesium
salts, or the like. When compounds contain relatively basic
functionalities, salts can be obtained by addition of the desired
acid, either neat or in a suitable inert solvent. Examples of
pharmaceutically acceptable acid addition salts include those
derived from inorganic acids like hydrochloric, hydrobromic,
nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galacturonic acids and the like (see, for
example, Berge et al, "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19).
The neutral forms of the compounds may be regenerated by contacting
the salt with a base or acid, and isolating the parent compound in
the conventional manner. The parent form of the compound differs
from the various salt forms in certain physical properties, such as
solubility in polar solvents, but otherwise the salts are
equivalent to the parent form of the compound for the purposes of
this invention.
In addition to salt forms, this invention provides compounds which
are in a prodrug form. Prodrugs of the compounds described herein
are those compounds that readily undergo chemical changes under
physiological conditions to provide the compounds of the present
invention. Additionally, prodrugs can be converted to the compounds
of the present invention by chemical or biochemical methods in an
ex vivo environment. For example, prodrugs can be slowly converted
to the compounds of the present invention when placed in a
transdermal patch reservoir with a suitable enzyme or chemical
reagent. Prodrugs are often useful because, in some situations,
they may be easier to administer than the parent drug. They may,
for instance, be more bioavailable by oral administration than the
parent drug. The prodrug may also have improved solubility in
pharmacological compositions over the parent drug. A wide variety
of prodrug derivatives are known in the art, such as those that
rely on hydrolytic cleavage or oxidative activation of the prodrug.
An example, without limitation, of a prodrug would be a compound of
the present invention which is administered as an ester (the
"prodrug"), but then is metabolically hydrolyzed to the carboxylic
acid, the active entity.
Certain compounds of the invention can exist in unsolvated forms as
well as solvated forms, including hydrated forms. In general, the
solvated forms are equivalent to unsolvated forms and are intended
to be encompassed within the scope of the present invention.
Certain compounds of the invention may exist in multiple
crystalline or amorphous forms. In general, all physical forms are
equivalent for the uses contemplated by the present invention and
are intended to be within the scope of the invention.
Some of the subject compounds possess asymmetric carbon atoms
(optical centers) or double bonds; the racemates, diastereomers,
geometric isomers and individual isomers are all intended to be
encompassed within the scope of the present invention.
The compounds are generally administered in a "therapeutically
effective amount", i.e. the amount of the subject compound that
will elicit the biological or medical response of a tissue, system,
animal or human that is being sought by the researcher,
veterinarian, medical doctor or other clinician. The term
"therapeutically effective amount" includes that amount of a
compound that, when administered, is sufficient to prevent
development of, or alleviate to some extent, one or more of the
symptoms of the condition or disorder being treated. The
therapeutically effective amount will vary depending on the
compound, the disease and its severity and the age, weight, etc.,
of the mammal to be treated.
The contacting is generally effected by administering to the
subject an effective amount of one or more compounds having the
general formula I (supra), including the various embodiments
described above. Generally administration is adjusted to achieve a
therapeutic dosage of about 0.1 to 50, preferably 0.5 to 10, more
preferably 1 to 10 mg/kg, though optimal dosages are compound
specific, and generally empirically determined for each
compound.
The term "unit dosage forms" refers to physically discrete units
suitable as unitary dosages for human subjects and other mammals,
each unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect, in
association with a suitable pharmaceutical excipient. Typical unit
dosage forms include prefilled, premeasured ampules or syringes of
the liquid compositions or pills, tablets, capsules, lozenges or
the like in the case of solid compositions. In such compositions,
the mimetic is usually a minor component (from about 0.1 to about
50% by weight or preferably from about 1 to about 40% by weight)
with the remainder being various vehicles or carriers and
processing aids helpful for forming the desired dosing form. Unit
dosage formulations are preferably about of 5, 10, 25, 50, 100,
250, 500, or 1,000 mg per unit. In a particular embodiment, unit
dosage forms are packaged in a multipack adapted for sequential
use, such as blisterpack comprising sheets of at least 6, 9 or 12
unit dosage forms.
It is understood that the examples and embodiments described herein
are for illustrative purposes only, not by way of limitation. Those
skilled in the art will readily recognize a variety of noncritical
parameters that could be changed or modified to yield essentially
similar results, and such modifications are included within the
spirit and purview of this application and scope of the appended
claims. All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes.
EXAMPLES: GENERAL EXPERIMENTAL
##STR00002##
Regent and Conditions: a) KOH MeOH, H.sub.2O, room temperature; b)
H.sub.2NNH.sub.2, MeOH, room temperature; c) pyridine,
CH.sub.2Cl.sub.2, room temperature.
##STR00003##
To a solution of methyl aryl ketone compound (1 eq) and KOH (3 eq)
in water and MeOH (water:MeOH=1:3) was added aryl carboxaldehyde
compound (1.05 eq) at room temperature. The solution was stirred at
room temperature for 10 hours, diluted with water, extracted with
ethyl acetate. The combined organic extracts was washed with brine,
dried over anhydrous Na.sub.2SO.sub.4, filtered, concentrated in
vacuo. The residue was purified by column chromatography (silica
gel, gradient elution, EtOAc:Hexane=1:5) to yield the desired
product alpha beta unsaturated ketone.
##STR00004##
To a solution of alpha beta unsaturated ketone compound (1 eq) in
MeOH was added hydrozine (3 eq) at room temperature. The solution
was stirred at room temperature for 10 hours, concentrated in
vacuo. The residue was washed with water and dried to yield the
desired pyrazole product that was used to next step without further
purification.
##STR00005##
To a solution of pyrazole compound (1 eq) in dichloromethane was
added corresponding acid chloride (1.1 eq) and pyridine (1.2 eq) at
room temperature. The solution was stirred for 6 hours at room
temperature, diluted with ethyl acetate, washed with brine, dried
over anhydrous Na.sub.2SO.sub.4, filtered, concentrated in vacuo.
The residue was purified by column chromatography (silica gel,
gradient elution, EtOAc:CH.sub.2Cl.sub.2=1:15) to yield the
product.
Activity is retained across the disclosed scope of embodiments of
formula, including the scope of R1, R2, R3, R4 and R5, as
exemplified in representative compounds show in the tables
below.
High Throughput Screens to identify Autophagy Inducers that
Function by Disrupting Beclin 1/Bcl-2 Binding
Autophagy, a lysosomal degradation pathway, plays a crucial role in
cellular homeostasis, development, immunity, tumor suppression,
metabolism, prevention of neurodegeneration and lifespan extension.
Thus, pharmacological stimulation of autophagy may be an effective
approach for preventing or treating certain human diseases and/or
aging. We sought to establish a method for developing new chemical
compounds that specifically induce autophagy. To do this, we
developed two assays to identify compounds that target a key
regulatory node of autophagy induction--specifically, the binding
of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an
allosteric modulator of the Beclin 1/VPS34 lipid kinase complex
that functions in autophagy initiation). These assays use either a
split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells
or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2
binding in vitro. We screened two different chemical compound
libraries, comprising .about.300K compounds, to identify small
molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy.
Three novel compounds were identified that directly inhibit Beclin
1/Bcl-2 interaction with an IC.sub.50 in the micromolar range and
increase autophagic flux. These compounds do not demonstrate
significant cytotoxicity and they exert selectivity for disruption
of Bcl-2 binding to the BH3 domain of Beclin 1 compared to the BH3
domain of the pro-apoptotic Bcl-2 family members, Bax and Bim.
Development of Beclin 1/Bcl-2 Split-Luciferase Assay
To identify novel compounds that disrupt Beclin 1/Bcl-2
interaction, we developed two new HTS assays designed in parallel
to (1) identify compounds that have cell penetration activity and
function in a cell-based assay to disrupt Beclin 1/Bcl-2
interaction; and (2) identify compounds that disrupt Beclin 1/Bcl-2
binding in vitro and therefore can be verified to function
on-target in the cell-based assay.
The first HTS employed a cell-based split-luciferase assay, which
is a proximity-based enzyme complementation reporter system. The
split-luciferase approach relies on the reconstitution of two
fragments of firefly luciferase, NLuc (amino acids 2-416) and CLuc
(amino acids 398-550) expressed with proteins of interest as fusion
partners. Upon the binding of interacting partners, two
non-functional fragments of the luciferase are brought into
proximity, forming an active luciferase protein. To measure Beclin
1/Bcl-2 interaction with the split-luciferase assay, we created
HeLa cell lines expressing N-terminal NLuc-tagged Beclin 1
(NLuc-Beclin 1) and CLuc-tagged Bcl-2 (CLuc-Bcl-2) as
split-luciferase reporters. Both reporters were expressed under the
control of a tetracycline-inducible promoter to avoid toxicity or
clonal adaptations that might potentially occur as a result of
constitutive overexpression of Beclin 1 or Bcl-2. Renilla
luciferase was constitutively expressed as an internal control. The
interaction of Beclin 1/Bcl-2 was measured as relative luminescence
units (RLU), which was the calculated ratio of split-luciferase and
Renilla luciferase signals:
.times..times..times..times..times..times..times..times..times..times..ti-
mes..times..times..times..times..times..times..times..times..times..times.-
.times..times..times..times..times. ##EQU00001##
The expression of Beclin 1 and Bcl-2 split-luciferase reporters in
HeLa cells yielded measurable luminescence activity that was
inhibited in a dose-dependent manner by the positive control
compound, ABT-737, a potent BH3 mimetic.sup.40. To confirm that
Beclin 1/Bcl-2 binding is required for the measured
split-luciferase activity, we also created cell lines expressing an
NLuc-Beclin 1 reporter carrying a deletion of the Bcl-2-binding
domain of Beclin 1 (lacking amino acids 81-151; referred to as
"Beclin 1.sup..DELTA.Bcl-2BD") and CLuc-Bcl-2. The expression of
Beclin 1.sup..DELTA.Bcl-2BD/Bcl-2 split-luciferase reporters
resulted in 10- to 20-fold reduction of luminescence activity and
the baseline activity of Beclin 1.sup..DELTA.Bcl-2BD/Bcl-2
split-luciferase reporters was not affected by ABT-737. Thus, the
full-length Beclin 1/Bcl-2 split-luciferase activity likely
represents a specific interaction between the BH3 domain of Beclin
1 and Bcl-2 rather than spontaneous re-association of luciferase
fragments. Overall, these data demonstrate that the HeLa cell
Beclin 1/Bcl-2 split-luciferase assay is robust and sensitive,
providing a wide dynamic range for measuring Beclin 1/Bcl-2
interaction.
We validated the Beclin 1/Bcl-2 split-luciferase assay for HTS with
a Z' factor test. The uniformity of the Beclin 1/Bcl-2
split-luciferase assay in a 384-well HTS format was examined. DMSO
(240 wells) and ABT-737 (16 wells) were used as neutral or positive
controls, respectively. The calculated Z' value was 0.7444,
indicating that Beclin/Bcl-2 split-luciferase assay is well-suited
for HTS.
Development of Beclin 1/Bcl-2 AlphaLISA Assay
We also developed a high-throughput Beclin 1/Bcl-2 AlphaLISA assay
to directly measure the in vitro interaction between Beclin 1 and
Bcl-2. AlphaLISA is a bead-based proximity assay that is capable of
measuring protein-protein interaction in homogeneous
solution..sup.41 Oxygen singlet molecules are generated by Alpha
donor beads upon irradiation at 680 nm and travel to acceptor beads
in proximity. Oxygen singlets excite donor beads and result in
luminescence emission at 615 nm. The half-life of oxygen singlet
molecules is extremely short such that efficient energy transfer
occurs only within a radius of 200 nm. Thus, a measurable Alpha
luminescence signal requires chemical energy transfer between the
donor and acceptor beads, and a pair of interacting molecules
immobilized on the beads stabilizes bead association to produce
measurable Alpha signals.
We used purified recombinant Beclin 1 and Bcl-2 proteins for
AlphaLISA, which were optimized for enhanced solubility and
expression. Human Beclin 1 was expressed as a fusion protein with
StrepII-SUMO at the N-terminus, and three residues on the aromatic
finger of Beclin 1 were mutated (F359D/F360D/W361D) to improve
solubility and protein stability..sup.42 These mutations are in the
BARA domain, which is far from the BH3 motif recognized by Bcl-2. A
recombinant Bcl-2-binding deficient mutant of Beclin 1
(StrepII-SUMO-Beclin 1.sup..DELTA.Bcl-2BD) was used as a negative
control. The Bcl-2 construct was composed of amino acids 1-218 and
truncated at the transmembrane domain for the addition of a
C-terminal 6.times.His tag (Bcl-2-6.times.His). For a normalization
control, we included a parallel AlphaLISA assay with purified SUMO
protein (with an N-terminal StrepII tag and a C-terminal
6.times.His tag). The purpose of a normalization control is to
eliminate inner filter (false-positive) hits that reduce
luminescence signal by interfering with the AlphaLISA assay in ways
that are unrelated to the Beclin 1/Bcl-2 interaction. With the
normalization control, the interaction of Beclin 1 and Bcl-2 was
measured as follows:
.times..times..times..times..times..times..times..times..times..times..ti-
mes..times..times..times..times..times. ##EQU00002##
The incubation of StrepII-SUMO-Beclin 1 and Bcl-2-6.times.His with
AlphaLISA beads produced a strong Alpha signal. Deletion of the
Bcl-2-binding domain of Beclin 1 almost completely abolished the
Alpha signal, indicating that the Alpha signal is specific to
Beclin 1/Bcl-2 interaction. Furthermore, the strong Alpha signal of
Beclin 1/Bcl-2, but not the weak Beclin 1.sup..DELTA.Bcl-2BD/Bcl-2
signal, was inhibited in a dose-dependent manner by the BH3
mimetic, ABT-737, which was used as a positive control for the
screen.
These data demonstrate that this assay is suitable for measuring
pharmacological inhibition of Beclin 1/Bcl-2 binding in vitro. To
further evaluate the Beclin 1/Bcl-2 AlphaLISA assay as an HTS
platform, we performed a Z' factor test using a 384-well plate
format. DMSO (240 wells) and ABT-737 (16 wells) were used as a
neutral or positive control, respectively. The resulting Z' value
was 0.7237, indicating that the Beclin 1/Bcl-2 AlphaLISA assay is a
robust HTS assay.
Primary and Secondary Screens for Disruptors of Beclin 1/Bcl-2
Binding
Using the Beclin 1/Bcl-2 split-luciferase assay, we performed a
primary HTS with chemical libraries comprising .about.200,000
compounds from UT Southwestern Medical Center (UTSW) and 100,000
compounds derived from diversity-oriented synthesis (DOS) at the
Broad Institute of MIT and Harvard..sup.43 The UTSW chemical
library is composed of 75,000 compounds purchased from ChemBridge
Corporation, 100,000 compounds purchased from Chemical Diversity
Labs, 22,000 compounds from ComGenex, 1200 purchased from TimTek,
1100 purchased from Prestwick, and 450 drugs from the NIH clinical
collection. Compounds purchased from TimTek are "natural
product-like" synthetic compounds, and the Prestwick compounds are
off-patent drugs. The NIH clinical collection is composed of
compounds that have been tested in phase I clinical trials. The
UTSW chemical library also contains approximately 30,000 natural
products isolated from unique marine bacteria by Dr. John MacMillan
(UC Santa Cruz). The compounds in the library satisfy a relaxed set
of Lipinsky's rules, with 99% having a molecular weight less than
550 g/mol (average 250-300 g/mol). All library compounds were
screened at a concentration of 5 .mu.M in a 384-well plate HTS
format. During screening, we noticed that a large number of library
compounds strongly increased Renilla Luciferase activity but did
not affect split-luciferase activity. Thus, to eliminate such
false-positive hits, we applied a Z-score cut-off of -3.0 on both
split-luciferase activity and normalized activity (RLU). Of the
identified 1027 hits from the UTSW library and 193 hits from the
Broad library, 233 and 55 hits were subsequently confirmed in a
repeat HTS assay, respectively.
To identify compounds that directly inhibit Beclin 1/Bcl-2
interaction, a selection of cherry-picked compounds (1027 from the
UTSW library and 55 from the Broad library) was subjected to a
secondary screen with the Beclin 1/Bcl-2 AlphLISA assay. In the
secondary HTS screen, we identified 35 (UTSW library) and one
(Broad library) compound which demonstrated >20% (UTSW library)
or >40% (Broad library) inhibition with a Z-score .ltoreq.-3.0.
We resupplied 19 compounds for additional dose-response AlphaLISA
(natural product fractions and compounds that were unavailable were
excluded). Six of the resupplied compounds were found to inhibit
Beclin 1/Bcl-2 interaction in vitro in a dose-dependent manner.
After removing pan-assay interference (PAINS).sup.44 compounds,
there were two compounds (SW063058 and SW076956) from the UTSW
library and one compound (BRD1991) from the Broad library chosen
for further hit validation and biological investigations.
Hit Validation and Assessment of Selectivity
We confirmed that these three candidate compounds showed a
dose-dependent inhibition of Beclin 1/Bcl-2 binding at an IC.sub.50
in the micromolar range using the AlphaLISA. To assess selectivity
for disruption of binding of the BH3 domain of Beclin 1 to Bcl-2 as
compared to that of a pro-apoptotic Bcl-2 family member, we used an
AlphaLISA with purified recombinant Bcl-2 and peptides spanning
either the BH3 domain (amino acids 105-130) of Beclin 1 or the BH3
domain (amino acids 49-84) of Bax. Our results indicate that all
three candidate molecules decreased the binding of the BH3 domain
of Beclin 1 but not the BH3 domain of Bax to Bcl-2. In contrast,
ABT-737 inhibited the binding of the BH3 domains of both Beclin 1
and Bax to Bcl-2 with nanomolar efficiency. Although it is still
possible that SW063058, SW076956 and BRD1991 might disrupt the
binding of Bcl-2 to Bax or other pro-apoptotic BH3
domain-containing molecules at a higher concentration (>20
.mu.M), these data clearly demonstrate a window for selective
inhibition of Beclin 1/Bcl-2 interaction.
For the two compounds for which we had sufficient supply for
large-scale tissue culture experiments (SW063058 and SW076956), we
examined their effects on Beclin 1, Bax, and Bim
co-immunoprecipitation with Bcl-2. Using previously described HeLa
cells that stably express Myc-tagged Bcl-2.sup.26, we observed that
12 h treatment with SW063058 or SW076956 decreased Beclin
1/Myc-Bcl-2 interaction, but not Bax/Myc-Bcl-2 or Bim/Myc-Bcl-2
interaction. The co-immunoprecipitation experiments in cells showed
the same trends as the AlphaLISA in vitro binding experiments;
SW076956 is more active than SW063058 in disrupting Beclin 1/Bcl-2
interaction (but not as active as the BH3 mimetic ABT-737) and
SW063058 and SW076956, but not ABT-737, selectively disrupt Beclin
1/Bcl-2 versus Bax/Bcl-2 binding. Moreover, ABT-737, but not
SW063058 or SW076956, disrupt co-immunoprecipitation between Bcl-2
and the BH3-only protein, Bim.
Functional Assays of Disruptors of Beclin 1/Bcl-2 Binding
The selective activity of SW063058, SW076956, and BRD1991 for
disrupting Beclin 1/Bcl-2 binding (versus Bax/Bcl-2 binding)
indicated that these compounds may induce autophagy without cell
death. We used well-established assays to assess their autophagic
activity,.sup.45 including (i) quantitation of GFP-LC3 puncta
(which label autophagosomes) in the presence and absence of the
lysosomal inhibitor, bafilomycin A1 (Baf A1), and (ii) western blot
analysis of the conversion of LC3-I to the lipidated,
autophagosomal-associated protein, LC3-II in the presence and
absence of Baf A1. HeLa cells stably expressing GFP-LC3 treated
with SW063058, SW076956, or BRD1991 had increased numbers of
GFP-LC3 puncta as compared to DMSO control 24 h after treatment
with 2 .mu.M compound. Shorter durations of lower concentrations of
compounds (10 .mu.M) also produced similar results. The numbers of
GFP-LC3 puncta increased further in the presence of Baf A1,
indicating that these compounds induce complete autophagic flux,
rather than a block in autophagolysosomal maturation. In support of
these findings, we also found that these compounds induce an
increase in LC3-II that further increases in the presence of Baf
A1, confirming an increase in autophagic flux. Thus, these
compounds induce autophagic flux in HeLa cells.
We assessed whether the induction of autophagy by SW063058 and
SW076956 requires Beclin 1 and Bcl-2 using siRNA to knockdown each
protein, respectively, in HeLa, cells. Consistent with its role as
an essential autophagy protein, Beclin 1 knockdown resulted in
decreased autophagosome numbers in baseline conditions (as
demonstrated by numbers of GFP-LC3 puncta) and, unlike in cells
subjected to control siRNA, there was no further increase in
GFP-LC3 puncta upon treatment with SW063058, SW076956 or ABT-737.
Consistent with its role as an autophagy inhibitor, Bcl-2 knockdown
resulted in increased numbers of GFP-LC3 puncta in baseline
conditions and, unlike in cells subjected to control siRNA, there
was no further increase in GFP-LC3 puncta upon treatment with
SW063058, SW076956 or ABT-737. We confirmed that Beclin 1 knockdown
decreased and Bcl-2 knockdown increased, respectively, autophagic
flux, by demonstrating increased levels of the autophagic
substrate, p62, in cells with Beclin 1 knockdown and decreased
levels of the autophagic substrate, p62, in cells with Bcl-2
knockdown. In contrast to cells treated with control siRNA,
SW063058, SW076956 or ABT-737 failed to alter p62 levels in cells
with either Beclin 1 or Bcl-2 knockdown. Taken together, these data
demonstrate that both Beclin 1 and Bcl-2 are required for the
autophagy-inducing effects of SW063058, SW076956 and ABT-737.
At the same concentrations that induce autophagic flux (10 and 20
.mu.M) as measured in the GFP-LC3 assay, SW063058 and SW076956 did
not exert cytotoxicity in HeLa cells whereas BRD1991 and ABT-737
exerted mild cytotoxicity at 20 .mu.M as measured by CelTiter-Glo
assays. Consistent with a previous report indicating that the
overexpression of Bcl-2 sensitizes cells to ABT-737-induced
apoptosis.sup.46, treatment with ABT-737, but not SW063058,
SW076956, or BRD1991, resulted in a more pronounced dose-dependent
increase in cell death in HeLa cells that overexpress Bcl-2. We
confirmed the CelTiter-Glo results using a more specific assay to
detect apoptotic death, the cleavage of the caspase 3 substrate,
PARP..sup.47 A dose-dependent increase in PARP cleavage was
observed with ABT-737 treatment, with cleaved PARP detected with 10
or 20 .mu.M compound. At these same concentrations, SW063058,
SW076956, and BRD1991 failed to induce PARP cleavage. Altogether,
these data indicate that there is a window in which our newly
identified disruptors of Beclin 1/Bcl-2 binding induce autophagy
without triggering apoptosis or other forms of cell death.
Pharmacokinetic Properties
Given the potential utility of these hits as starting points for
further SAR to develop specific autophagy inducers, we analyzed and
quantified cell penetration and measured some metabolic stability
properties. Using mass spectrometry, we measured the intracellular
drug concentration in HeLa cells at different time points after the
incubation with 1 .mu.M of either SW063058, SW076956 or BRD1991.
All three candidate compounds enter the cell within one hour, but
only SW076956 and BRD1991 showed gradual intracellular accumulation
over time. In measuring in vitro ADME properties for these hit
compounds, microscale thermodynamic solubility showed that the
compounds had solubility ranging from 0.72 .mu.M to 30 .mu.M in PBS
and 43.3 .mu.M to 202 .mu.M in AlphaLISA buffer. Plasma stability
was greater than 90% in murine and human plasma. Murine and human
microsomal stability showed that 36% of parental compound SW063058
was unmetabolized after one hour of human microsome extract
exposure while all other combinations of compounds exposed to
murine or human microsomes were present at concentrations of less
2% of parental compounds after 1 h. Depending on the scaffold
chosen to elaborate, these properties will need to be taken into
account in moving a series forward.
NMR Chemical Shift Perturbation Analysis
To examine whether SW063058, SW076956 and BRD1991 occupy the
hydrophobic pocket of Bcl-2, we carried out a chemical shift
perturbation analysis. The incubation of purified Bcl-2/-xL chimera
protein (in which the unstructured loop of Bcl-2 was replaced with
a short loop from Bcl-xL).sup.41 with the candidate molecules
resulted in chemical shift changes for several residues, indicating
that the candidate molecules bind to Bcl-2. Notably, a chemical
shift change was observed for F153 located inside the P2
hydrophobic binding pocket of Bcl-2, indicating that these
candidate compounds can bind within the BH3 pocket. Other chemical
shift changes of residues not in the vicinity of the P2 pocket
(G141, V162, and D171) is indicative of allosteric conformational
changes of Bcl-2/-xL chimera upon ligand binding. Overall, the
chemical shift profiles of SW063058, SW076956 and BRD1991 are
similar but showed some distinct features, as compared to known BH3
mimetics, including ABT-737 and ABT-199 and ABT-263. For example,
while SW063058, SW076956 and BRD1991 all resulted in a chemical
shift in F153 inside the hydrophobic groove (as did the BH3
mimetics), they did not result in chemical shifts in other residues
in the groove such as F104 and E136; in addition, SW063058,
SW076956 and BRD1991 led to chemical shifts in G155, a residue
essential for Bcl-2 binding to Beclin 1,.sup.49 whereas ATB-737 and
other BH3 mimetics did not. These data indicate that the structural
determinants for the binding of Bcl-2 to our newly identified
Beclin 1/Bcl-2 binding disruptors is different from that of known
BH3 mimetics.
We have developed two new HTS approaches for identifying compounds
that disrupt the Beclin 1/Bcl-2 interaction. These screening tools
resulted in the identification of three validated hits, SW063058,
SW076956 and BRD1991, that selectively disrupt Beclin 1/Bcl-2
binding as compared to Bax/Bcl-2 and Bim/Bcl-2 binding and induce
autophagic flux at concentrations that showed minimal cytotoxicity.
Moreover, NMR chemical shift perturbation data indicate that these
newly identified compounds can bind to the P2 pocket of Bcl-2 via a
modality that is distinct from currently available BH3
mimetics.
A previous screen was recently reported with a smaller number of
compounds to identify small molecules that function as Beclin 1 BH3
mimetics and induce both autophagy and apoptosis..sup.50 In
contrast, our goal was to identify compounds that only induce
autophagy by exerting selectivity for disrupting the binding of the
BH3 domain of Beclin 1 but not the BH3 domain of Bax to Bcl-2. We
identified three initial tool compounds that meet these criteria.
Although they are biologically active in the micromolar range, the
properties identified in this study--especially their selectivity
for disrupting the interaction between Bcl-2 and the Beclin 1 BH3
motif, their ability to induce autophagy without cytotoxicity, and
their unique NMR chemical shift perturbation profiles (compared to
known BH3 mimetics)--rendered them strong hit compounds for design
of analogs of increasing potency, increasing selectivity for
disrupting Beclin 1/Bcl-2 versus Beclin 1/Bax (or other
pro-apoptotic BH3-containing proteins) interaction, and optimizing
ADME properties. The cell-based and in vitro based Beclin 1/Bcl-2
binding assays provides a useful both for rapid
assessment/optimization of such analogs.
We used these compounds as starting points for the development of
effective Beclin 1/Bcl-2 selective inhibitors, and our
structure-activity relationship work produced autophagy modulators
further described herein.
Cell Lines
HeLa/GFP-LC3.sup.51 and HeLa/Myc-Bcl-2.sup.26 cells were previously
described by our laboratory. Beclin 1/Bcl-2 split-luciferase cells
were generated by stable transfection of HeLa tet-on cells
(Clontech) with the tetracycline-inducible plasmid pTRE2pur vector
expressing NLuc-Beclin 1 (or Beclin 1.sup..DELTA.Bcl-2BD mutant)
and the pTRE2-RLuc-hyg3 vector expressing CLuc-Bcl-2. Stable clones
were selected in selection medium (DMEM supplemented with DMEM
supplemented with 10% Tet-system-approved FBS, 1 mM glutamine, 100
.mu.g/mL G418, 200 .mu.g/mL hygromycin, and 0.5 .mu.g/mL
puromycin). The previously described Beclin 1.sup..DELTA.Bcl-2BD
mutant.sup.24, 49 contains a deletion in the Beclin 1 BH3 domain
spanning from amino acids 88-150 which interferes with its binding
to Bcl-2.
Recombinant Proteins and Peptides
The human beclin 1 gene was cloned into the expression vector
ppSUMO-Strep as a fusion with N-terminal StrepII tag and SUMO
protein. Three aromatic residues located at the "aromatic finger"
of human Beclin 1.sup.42 were mutated to aspartate
(F359D/F360D/W361D) to improve protein solubility. E. coli BL21
Star (DE3) pLysS containing StrepII-SUMO-Beclin 1,
StrepII-SUMO-Beclin 1.sup..DELTA.Bcl-2BD or
StrepII-SUMO-6.times.His expression plasmids were grown at
37.degree. C. until logarithmic phase and incubated in 0.1 mM IPTG
at 16.degree. C. overnight. Soluble StrepII-SUMO-Beclin 1 or
StrepII-SUMO-Beclin 1.sup..DELTA.Bcl-2BD protein was
affinity-purified by StrepTactin sepharose (IBA Biosciences) and
further purified by size exclusion chromatography with a HiLoad
16/60 Superdex 200 column (GE Healthcare) equilibrated with
Modified Buffer W (100 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5%
glycerol, 0.1% CHAPS, and 1 mM DTT).
For the purification of Bcl-2, the expression plasmid pET21c-Bcl-2
(spanning amino acid 1-218), which encodes human Bcl-2 lacking the
transmembrane domain with a C-terminal 6.times.His tag was used.
BL21 Star (DE3) pLysS harboring the expression plasmid was grown at
37.degree. C. until logarithmic phase, and IPTG was added to a
final concentration of 0.1 mM and incubated at 16.degree. C.
overnight. Soluble Bcl-2-6.times.His protein was affinity purified
by Ni-NTA agarose (Qiagen) with lysis buffer (50 mM phosphate, pH
8.0, 300 mM NaCl, 0.1% CHAPS and 5% glycerol). Imidazole was used
at 10 mM for binding, 20 mM for washing, and 250 mM for elution.
The affinity purified Bcl-2-6.times.His was further purified by
size exclusion chromatography with a HiLoad 16/60 Superdex 200
column equilibrated with Modified Buffer W.
NMR experiments used a chimeric version (Bcl-2/-xL) of Bcl-2 that
substitutes the Bcl-xL loop to improve the 2D spectra..sup.48 DNA
was optimized for expression in E. coli (GeneArt, Life
Technologies) and cloned into a pET28a vector with an N-terminal
His.sub.6-MBP tag to aid overexpression. A TEV cleavage site was
engineered between the MBP tag and Bcl-2/-xL proteins for removal
of the MBP-tag. The protein was expressed and labeled as
described.sup.52 and protein purification was done as
described..sup.53 Protein purity was estimated to be .gtoreq.95% by
SDS-PAGE, with image analysis by ImageJ, and quantified using an
.epsilon..sub.280 of 43,430 M.sup.-1 cm.sup.-1. Aliquots were
concentrated to 100 .mu.M and frozen at -80.degree. C. until
needed.
The Beclin 1 BH3 peptide consists of an N-terminally linked biotin,
a YGGGGS linker, and 16 amino acids (a.a. 105-130) derived from
human Beclin 1. Bax BH3 peptide consists of an N-terminally linked
biotin, a YGGGGS linker, and 36 amino acids (a.a. 49-84) derived
from human Bax. Biotin-6.times.His control peptide consists of an
N-terminally linked biotin, a YGGGGS linker, and a 6.times.His tag
at C-terminus.
Beclin 1/Bcl-2 Split-Luciferase Screen
Beclin 1/Bcl-2 split-luciferase cells were suspended in induction
medium (DMEM supplemented with Tet-approved FBS, 1 mM glutamine,
100 units/mL of Penicillin/Streptomycin, and 1 .mu.g/mL
doxycycline) and plated at 1.2.times.10.sup.4 cells/well in
384-well plates (Corning #3570) and incubated at 37.degree. C.
overnight.
DMSO (neutral control), ABT-737 (positive control), or the library
compounds were added to a final concentration of 5 .mu.M and
incubated at 37.degree. C. Compounds were from either the UTSW
(200K) or the Broad Institute of MIT and Harvard diversity-oriented
synthesis (100K) collection (provided courtesy of Stuart
Schreiber). After 5 h of incubation, the medium was removed by
centrifugation and the activity of Beclin 1/Bcl-2 split-luciferase
reporters was measured with Dual-Glo.RTM. Luciferase Assay System
(Promega). Twenty microliters of 1.times.FL buffer (1:1 dilution of
Luciferase Reagent with PBS) was first added to the well, incubated
at room temperature for 10 min, and firefly luminescence units were
measured by an Envision plate reader (Perkin Elmer). Ten
microliters of 1.times.RL buffer (Stop & Glo Reagent) was then
added to the sample, the sample was incubated at room temperature
for 10 min, and the activity of Renilla luciferase was measured
again. The Beclin 1/Bcl-2 interaction was measured as relative
luminescence units (RLU), calculated as the ratio of
split-luciferase and Renilla luciferase signals.
The screen was performed in a 384-well format. Each assay plate
contained 320 library compounds in column 3 to 22. DMSO was
included as a neutral control in column 2 and 23, and ABT-737 was
used as a positive control in column 1. For analysis of the primary
screen data, numerical readouts obtained from EnVision plate reader
were quality controlled and processed using the Assay Analyzer
module of the Genedata Screener.RTM. Suite. To remove systemic
variation bias such as edge effect or plate effect, normalized
values (RLU) were corrected with a proprietary pattern detection
algorithm in the Assay Analyzer software..sup.54 Z-scores were
calculated from the split-luciferase activity and the corrected
normalized activity (RLU) for each compound..sup.54 The percent
activity of a compound at the tested concentration was defined
as:
.times..times..times..times..times..times..times..times.
##EQU00003##
Compounds having Z-scores less than -3.0 in the split-luciferase
activity and the corrected normalized activity (RLU) were advanced
for study in the confirmation assays.
The confirmation assay for compounds from the UTSW library was
performed in triplicate using compound concentrations of 5 .mu.M.
The confirmation assays for compounds from the Broad Institute
library were performed at 5 .mu.M with a 2-fold 8-point serial
dilution. The values of percent activity in the triplicates for
each compound were then condensed to a single value as "condensed
activity", the representative single value of the triplicates,
using the "Robust Condensing" method in Genedata Screener.RTM.. In
general, the triplicates were pre-condensed into a pair of values
(X and Y) as follows: Values (X,Y)=(Median of Triplicates
m).+-.Dispersion Dispersion=Median
(|X.sub.1-m|,|X.sub.2-m|,|X.sub.3-m|)
A lower |X-Y| value indicated better data quality. For data points
where |X-Y|.ltoreq.30%, the median of X and Y was used as the
condensed activity, which is also the median of the triplicate
measurements. Otherwise, a condensing function Max (X,Y) was used
to estimate the condensed activity. A robust Z-Score was then
calculated for each compound using the following equation:
.times..times..times..times..times..times..times..times..times..times..ti-
mes..times..times..times..times..times..times..times..times..times..times.-
.times..times..times..times..times. ##EQU00004##
Compounds having Z-scores less than -3.0 (UTSW library) or
dose-response inhibition of split-luciferase activity (Broad
Institute library) were considered confirmed.
Beclin 1/Bcl-2 AlphaLISA Screen
All AlphaLISA assays were performed in triplicate in a 384-well
format with BB buffer (phosphate-buffered saline supplemented with
0.5% BSA and 1 mM DTT). For the Beclin 1/Bcl-2 AlphaLISA, purified
StrepII-SUMO-Beclin 1 was incubated with Bcl-2-6.times.His proteins
at 300 nM and 60 nM, respectively. For internal control AlphaLISA
assays, StrepII-SUMO-6.times.His protein (internal control) was
used at 20 nM. Samples containing Beclin 1/Bcl-2 or SUMO proteins
were added to AlphaPlates-384 (Perkin Elmer #6005350) by Multidrop
and incubated with DMSO (neutral control), ABT-737 (positive
control), or library compound at 5 .mu.M (UTSW library) or 10 .mu.M
(Broad library) at room temperature for 3 h to allow
protein-protein interaction. Following the initial incubation,
Strep-Tactin Alpha donor (AS106D) and anti-6.times.His AlphaLISA
acceptor beads (AL128M) were added to a final concentration of 40
.mu.g/mL each and incubated at room temperature in the dark for an
additional 1 h. All samples were evaluated in triplicate. Alpha
signals were measured by an Envision plate reader (Perkin Elmer).
Beclin 1/Bcl-2 Alpha signal was normalized with internal control
Alpha signal. The normalized value represents the binding activity
of Beclin 1 and Bcl-2. The percent activity of a compound was
calculated as the following formula:
.times..times..times..times..times..times..times..times..times..times..ti-
mes..times..times..times..times..times..times..times..times..times..times.-
.times..times..times. ##EQU00005##
The values of percent activity of each compound in triplicates were
converted into condensed activity and a robust Z-score was
calculated as described in the previous section. Compounds with a Z
score less than -3.0 and percent activity >20% (UTSW library) or
>40% (Broad Institute library) and were selected for further
confirmation with dose-response experiments.
For dose-response AlphaLISA experiments, compounds were serially
diluted (2-fold, 7 point) with DMSO and added to BB buffer
containing purified recombinant proteins or biotinylated peptides
at a final concentration range of 10 .mu.M to 156 nM. Protein or
peptide concentrations used in dose-response AlphaLISA experiments
were as follows: Beclin1/Bcl-2-300 nM/60 nM; SUMO (internal control
for Beclin 1/Bcl-2 AlphaLISA)--20 nM; Beclin 1 BH3/Bcl-2--1000
nM/60 nM; Bax BH3/Bcl-2--100 nM/20 nM; Biotin-6.times.His (internal
control for BH3/Bcl-2 AlphaLISA)--10
Analytical Assays
Solubility was either measured in PBS buffer by the Analytical
Group of the Broad Institute or in AlphaLISA reaction buffer by the
Preclinical Pharmacology Core Laboratory at UT Southwestern Medical
Center. To measure solubility in PBS buffer, each compound was
prepared in triplicate at 100 .mu.M in both 100% DMSO and PBS with
1% DMSO. Compounds were allowed to equilibrate at room temperature
with a 750 rpm vortex shake for 18 h. After equilibration, samples
were analyzed by UPLC-MS (Waters, Milford, Mass.) with compounds
detected by SIR detection on a single quadrupole mass spectrometer.
The DMSO samples were used to create a two-point calibration curve
to which the response in PBS was fit. To measure solubility in
AlphaLISA reaction buffer, each compound was prepared in triplicate
at 1 mM in PBS containing 0.5% BSA and 1 mM DTT in glass vials. The
vials were shaken vigorously (250 rpm) on an orbital shaker for 18
h. The samples were placed in teflon eppendorf tubes and
centrifuged at 16,100.times.g for 10 min. The supernatant was
collected and analyzed by a Qtrap 3200 LC-MS/MS system.
Plasma stability was determined at 37.degree. C. at 5 h in both
human and mouse plasma. Each compound was prepared in duplicate at
5 .mu.M in plasma diluted 50/50 (v/v) with PBS pH 7.4 (0.95%
acetonitrile, 0.05% DMSO). Compounds were incubated at 37.degree.
C. for 5 h with a 350-rpm orbital shake with time points taken at 0
h and 5 h. Samples were analyzed by UPLC-MS (Waters, Milford,
Mass.) with compounds detected by SIR detection on a single
quadrupole mass spectrometer.
Microsomal stability was determined at 37.degree. C. at 60 min in
both human and mouse microsomes. Each compound was prepared in
duplicate at 1 .mu.M with 0.3 mg/mL microsomes in PBS pH 7.4 (1%
DMSO). Compounds were incubated at 37.degree. C. for 60 min with a
350-rpm orbital shake with time points taken at 0 min and 60 min.
Samples were analyzed by UPLC-MS (Waters, Milford, Mass.) with
compounds detected by SIR detection on a single quadrupole mass
spectrometer.
NMR Spectroscopy Experiments
The NMR data were acquired using a Bruker Avance III HD 600 MHz
spectrometer equipped with a cryogenic QCI cryoprobe at the Broad
Institute. The 2D .sup.15N-.sup.1H HSQC and TROSY spectra were
collected at 25.degree. C. in the absence or presence of compound.
The NMR samples were comprised of 90% H.sub.2O/10% D.sub.2O in 25
mM HEPES (pH 7.5), 150 mM NaCl and 0.5 mM TCEP. For the chemical
shift perturbation experiments with ABT-737, ABT-199, ABT-263,
SW063058, SW076956 and BRD1991, aliquots were titrated into the
.sup.15N-labeled Bcl-2/-xL and 2D .sup.5N-.sup.1H HSQC and TROSY
spectra were collected at 25.degree. C. The chemical shift
assignments for Bcl-2/-xL were performed as previously
described..sup.48 All of the NMR data were analyzed using SPARKY
software.
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TABLE-US-00001 TABLE 1 Active compounds ID Structure IC50 (uM) Log
P ClogP CLogD tPSA 1qr-8-175 ##STR00006## N/A -0.27 1.582 0.62
79.79 1qr-8-177 ##STR00007## 16.17 2.07 3.076 2.7 88.07 1qr-8-178
##STR00008## N/A -0.32 1.142 0.33 88.07 1qr-8-179 ##STR00009## N/A
1.66 3.173 2.45 88.07 1qr-8-180 ##STR00010## 2.765 2.14 3.29 2.79
88.07 1qr-8-181 ##STR00011## N/A 1.58 2.577 2.19 88.07 1qr-8-182
##STR00012## 18.24 2.07 3.076 2.7 88.07 1qr-8-183 ##STR00013##
12.84 1.74 2.72 2.33 88.07 1qr-8-184 ##STR00014## N/A 1.74 2.72
2.33 88.07 1qr-8-185 ##STR00015## 9.895 1.45 2.496 2.03 97.3
1qr-8-186 ##STR00016## 2.421 2.41 3.44 2.96 88.07 1qr-8-187
##STR00017## N/A NA 2.68 2.46 105.14 1qr-8-188 ##STR00018## 1.676
1.61 2.32 2.13 139.88 1qr-8-189 ##STR00019## 2.794 2.5 3.46 3.07
88.07 1qr-8-190 ##STR00020## 7.227 1.61 2.01 2.04 111.86 1qr-8-192
##STR00021## N/A 0.2 1.753 2.28 97.3 1qr-8-194 ##STR00022## N/A
2.19 4.909 2.8 71 1qr-8-195 ##STR00023## N/A 3.25 4.716 4 88.07
1qr-8-196 ##STR00024## 2.14 2.94 3.7 3.12 88.07 1qr-8-176
##STR00025## N/A 4.4 4.81 5.06 96.35 1qr-8-203 ##STR00026## 2.879
4.21 4.973 5.24 80.23 1qr-8-205 ##STR00027## N/A 3.04 3.04 3.48
67.92 1qr-8-211 ##STR00028## N/A 4.74 5.204 5.31 88.07 1qr-8-212
##STR00029## N/A 2.75 3.286 3.57 71 1qr-8-214 ##STR00030## N/A 4.24
5.185 3.87 88.07 1qr-8-219 ##STR00031## 29.78 3.68 4.295 4.13 53.93
1qr-8-228 ##STR00032## N/A 3.88 4.01 4.27 93.71 1qr-8-229
##STR00033## N/A 3.72 4.186 4.17 51.13 1qr-8-230 ##STR00034## N/A 4
4.41 4.47 41.9 1qr-8-235 ##STR00035## 1.801 3.34 3.736 4.19 80.23
1qr-8-236 ##STR00036## N/A 4.32 5.135 5.47 83.03 1qr-8-239
##STR00037## N/A 4.63 4.71 4.63 90.66 1qr-8-240 ##STR00038## N/A
2.9 3.652 3.63 86.6 1qr-8-241 ##STR00039## N/A 5.48 5.67 5.75 69.86
1qr-8-242 ##STR00040## N/A 3.14 3.483 3.85 77.81 1qr-8-231
##STR00041## 0.9822 4.56 5.337 5.49 80.23 1qr-8-232 ##STR00042##
N/A 4.56 5.337 5.49 80.23 1qr-8-233 ##STR00043## 1.244 3.76 4.217
4.67 132.04 1qr-8-238 ##STR00044## 0.9048 3.45 3.9 3.64 78.84
1qr-8-267 ##STR00045## 13.92 4.28 4.763 4.41 78.84 1qr-8-268
##STR00046## N/A 4.28 4.763 4.41 78.84 1qr-8-269 ##STR00047## 9.83
4.28 4.763 4.41 78.84 1qr-8-275 ##STR00048## 14.16 3.94 4.399 4.15
78.84 1qr-8-276 ##STR00049## N/A 3.49 3.643 3.58 130.65 1qr-8-278
##STR00050## 0.2741 5.94 6.161 6.43 71 1qr-8-279 ##STR00051##
0.3493 5.14 5.041 5.61 122.81 Lqr-8-296 ##STR00052## >10 4.43
4.02 4.1 32.67 1qr-8-302 ##STR00053## 2.68 5.26 6.2 5.96 80.23
1qr-8-309 ##STR00054## >10 4.19 4.63 4.58 41.9 1qr-8-310
##STR00055## >10 4.12 4.06 4.52 44.7 1qr-9-004 ##STR00056##
>10 3.82 3.66 4.51 105.14 1qr-9-008 ##STR00057## 10 5.07 4.92
5.38 71 1qr-9-012 ##STR00058## >10 4.33 5.27 4.79 62.72
1qr-9-013 ##STR00059## >10 6.73 8.76 6.73 71 1qr-9-018
##STR00060## >10 5.52 5.75 5.52 62.21 1qr-9-026 ##STR00061## 13
5.03 5.45 5.59 71 1qr-9-033 ##STR00062## 0.455 5.03 4.66 5.45 83.36
1qr-9-044 ##STR00063## 1.8 4.6 4.66 5.22 83.36 1qr-9-046
##STR00064## 3 4.6 4.66 5.21 83.36 1qr-9-048 ##STR00065## 4.1 4.5
5.33 5.57 80.23 1qr-9-055 ##STR00066## 12 5.94 6.16 6.43 71
1qr-9-056 ##STR00067## 10 4.67 5.53 5.48 80.23 Lqr-9-065
##STR00068## 6.77 4.89 5.26 5.29 89.46 1qr-9-073 ##STR00069##
>10 5.57 5.29 6.36 88.07 1qr-9-076 ##STR00070## 0.3494 5.04 5.25
5.42 73.8 1qr-9-085 ##STR00071## 0.063 5.72 5.76 6.15 71 1qr-9-088
##STR00072## 5.138 5.06 4.46 5.07 62.21 1qr-9-118 ##STR00073## 9.1
5.26 5.65 5.71 73.8 1qr-9-121 ##STR00074## 5.52 4.39 4.41 4.65 73.8
1qr-9-136 ##STR00075## 7.5 4.42 4.74 5.22 88.07 1qr-9-068
##STR00076## >10 5.77 5.43 62.1 1qr-9-155 ##STR00077## 0.184
6.29 6.38 6.62 71 1qr-9-156 ##STR00078## <0.156 5.76 5.7 6.11 71
1qr-9-157 ##STR00079## <0.156 5.4 5.45 5.74 71 1qr-9-158
##STR00080## 2.39 4.62 4.29 4.87 65.01 1qr-9-161 ##STR00081##
<0.156 5.36 5.51 5.85 71 1qr-9-163 ##STR00082## >10 4.94 4.6
5.28 65.01
TABLE-US-00002 TABLE 2 SAR: activity is retained across R1 scope
##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087##
##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092##
##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097##
##STR00098##
TABLE-US-00003 TABLE 3 SAR: activity is retained across R3 scope
##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103##
##STR00104## ##STR00105## ##STR00106##
TABLE-US-00004 TABLE 4 SAR: activity is retained across R4 scope
##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111##
##STR00112##
* * * * *